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Fatty acid composition of subcutaneous and intramuscular adipose tissues and M. longissimus dorsi of Wagyu cattle
Meat Science 32 (1992) 449-458 t'411Ji'IJ
Fatty Acid Composition of Subcutaneous and Intramuscular Adipose Tissues and M. longissimus dorsi of Wagyu Cattle C. A. Sturdivant, D. K. Lunt, G. C. Smith & S. B. Smith* Department of Animal Science, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843, USA (Received 30 June 1991; revised version received 22 November 1991; accepted 24 November 1991) ABSTRACT Three experiments were conducted to document the fatty acid composition of tissues from purebred Wagyu cattle from Japan and North American crossbred Wagyu. In experiment 1, subcutaneous (s.c.) adipose tissues ( n = 23) were obtained from Japanese cattle representing the.17veJapanese fat quality grades. The monounsaturated:saturated fotty acid ratio (MUFA:SFA ) was greatest in.fat quality grade 5 samples (2.5 7) and least in thefat quality grade 3 samples (2.08: P < 0"05). In experiment 2, M. longissimus dorsi and the associated intramuscular ( i.m.) and s.c. adipose tissues were obtahled /)'om carcasses of Wagyu crossbred steers (1/2-7/8) raised in the USA. Fatty acid composition varied among depots, but the MUFA:SFA ratio in s.c. adipose tissue (1"46) was not d(fferent from values reported for other breeds' o['cattlc. In experiment 3, samples of M. longissimus dorsi ribsteaks were obtained from three regions in Japan. Samples from the Gunma region had the greatest ( P < 0"05) M UFA :SFA ratio (2"10), relative to samples /i'~m the Kagoshima (1.82) and Miyazaki (1"65) regions. The data indicate that bee/ from purebred Wagyu cattle raised in Japan is enriched in monounsaturated fatty acids, and that the degree of enrichment depends upon the region of Japan from which the samples were obtained. INTRODUCTION Because o f the difficulties in modifying the fatty acid composition of bovine tissues by dietary means (e.g. Smith, 1991), selection of cattle with a greater * To whom correspondence should be addressed. 449 Meat Science0309-1740/92/$05.00 © 1992 ElsevierScience Publishers Ltd, England. Printed in Great Britain
450
C. A. Sturdivant, D. K. Lunt, G. C. Smith, S. B. Smith
genetic propensity to deposit specific fatty acids may be the primary means by which fatty acid composition is modified in ruminant species. Breed comparisons of fatty acid composition are difficult because of the confounding of age, plane of nutrition and other extrinsic factors (Yoshimura & Namikawa, 1983; Eichorn et al., 1986). In a recent investigation in which these factors were controlled, s.c. adipose tissue from mature Bos indicus cows contained greater percentages of the monounsaturated fatty acids myristoleate (14:1), palmitoleate (16:1) and oleate (18:1) than the adipose tissue from Bos taurus cows (Huerta-Leidenz et al., in press). This resulted in a greater ratio of monounsaturated:saturated (MUFA: SFA) fatty acids in adipose tissue from the Bos indicus cows (1.85 vs 1.54 for the Bos taurus cows). Although the differences in the individual fatty acids were not great (e.g. 49-6% oleate in Bos indicus adipose tissue vs 47.7% oleate in the Bos taurus adipose tissue), the results of Huerta-Leidenz et al. (in press) suggested that genetic differences do exist in the ability of cattle to synthesize and deposit monounsaturated fatty acids. Tanaka (1985) reported that s.c. adipose tissue from Japanese Black (Wagyu) steers contained over 48% oleate and had an M U F A :SFA ratio of approximately 1.5, compared with an M U F A : S F A ratio of 1.2 for adipose tissue from Japanese Shorthorn and Holstein steers. The objectives of this research were to provide additional information about the genetic basis for differences in fatty acid composition among breeds of cattle, and to further characterize the Wagyu breed, first by documenting fatty acid compositional differences among Japanese fat quality grades. The initial interest in evaluating differences among fat quality grades was generated by the subjective observation that increases in fat quality grade (which are ranked from 1 to 5) were associated with softer s.c. adipose tissue on chilled carcasses. The extraordinarily high concentrations of monounsaturated fatty acids in s.c. adipose tissue from purebred Black Wagyu cattle from Japan led to investigations of tissues from crossbred Wagyu steers raised in the USA. This was followed by investigations of regional differences in fatty acid composition.
MATERIALS AND METHODS
Experimental design Experiment 1
Twenty-three samples of s.c. adipose tissue were obtained from a commercial meat processing plant in Japan and transported on dry ice to the Department of Animal Science at Texas A & M University. Samples were
Fatty acid composition of Wagyu tissues
451
obtained at slaughter from Japanese Black Wagyu steers representing the five Japanese fat quality grades. Japanese Black Wagyu steers typically are slaughtered at 30-34 months of age. Samples were held frozen at - 2 0 ° C until analyzed for fatty acid composition.
Experiment 2 One-half to 7/8 Wagyu crossbred steers (n = 8) were fed a corn-milo feedlot ration free choice from a covered self-feeder for 174 days and then fed a corn-based feedlot ration until time of slaughter. Both Black and Red Wagyu bulls were mated to Angus and Hereford × Angus cows to produce the steers sampled in this experiment. Cattle were slaughtered when backfat thickness reached 1"8 cm, as determined by ultrasonography. The steers were approximately 27 months of age at slaughter. After the carcasses had been fabricated, samples of s.c. adipose tissue, M. longissimus dorsi and i.m. (marbling) adipose tissue from within the muscle were obtained by dissection. Samples were held frozen at - 2 0 ° C until analyzed for fatty acid composition. Experiment 3 The USA recently has allowed importation of Japanese beef. Eleven strip steaks from Japanese Black Wagyu steers produced and slaughtered in Japan were obtained from outlets in the USA. The steaks came from cattle representing three different regions of Japan: the Kagoshima region (n = 5), the Miyazaki region (n = 4) and the Gunma region (n = 2). From each steak, s.c. adipose tissue, M. longissimus dorsi, and i.m. adipose tissue from within the muscle were obtained by dissection. For subsequent analyses, the raw s.c. adipose tissue, muscle, and marbling samples were stored at -20°C.
Fatty acid analysis Fatty acids were determined by gas chromatography as described by HuertaLeidenz et al. (1991). An aliquot of the total lipid extract from each of the samples was freed of solvent under nitrogen using an analytical evaporator (Meyer N-Evap, Organomation Associates Inc., Berlin, MA). Each volume of the aliquot was calculated to yield approximately 25 mg of total lipid extract from the longissimus dorsi muscle. The extract was methylated with boron trifluoride-methanol following the procedure described by Morrison & Smith (1964). Fatty acid methyl esters (FAME) were analyzed using a flame ionization gas chromatograph (Packard, Model 437A, Chrompack Inc., Raritan, N J) equipped with a 2 m × 3"175 mm stainless steel column packed with 15% cyanopropylphenylpolysiloxane (9:1w/w) on a 100-120 mesh solid support. The column was run isothermally at a temperature of 180°C.
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C. A. Sturdivant, D. K. Lunt, G. C. Smith, S. B. Smith
F A M E in 1/A o f h e x a n e was delivered into the column using a microsyringe. The injection port and detector were maintained at 250°C. Flow rates were 16ml/min for the carrier gas (nitrogen), 34ml/min for hydrogen, and 214ml/min for breathing air. C h r o m a t o g r a m s were recorded with a computing integrator (Spectraphysics Model SP4290, San Jose, CA). The gas c h r o m a t o g r a p h system was calibrated with standard F A M E mixtures (Nu-Chek Prep. Inc., Elysian, MN). Identification o f sample fatty acids was made by comparing the relative retention times of F A M E peaks from samples with those of standards.
Statistical analysis Data were analyzed with the General Linear Program (SAS, 1986). Experiments 1 and 2 were analyzed by one-way analysis o f variance (Ott, 1984). F o r experiment 1, the main effect tested was fat quality grade. F o r experiment 2, the main effect tested was depot site. Experiment 3 was analyzed as a 3 × 3 factorial design (region x tissue site). The main effects analyzed were depot site and regions within Japan, and the region x tissue site interaction was tested. The analysis o f variance was used to indicate significant differences (P < 0"05) a m o n g means. When indicated by analysis o f variance, means were separated by the S t u d e n t - N e w m a n - K e u l s method (Ott, 1984).
TABLE 1 Percentages of Major Fatty Acids in Subcutaneous Adipose Tissue Samples of Wagyu Cattle as Affected by Japanese Fat Quality Grade (Experiment 1)a Fatty acid
14:0 14:1 16:0 16:1 18:0 18:1 18:2 MUFA:SFAb
Quality grade
C.V.
1 (n=5)
2 (n=4)
3 (n=4)
4 (n=6)
5 (n=4)
3"19 2"70 22.99 11'32 4'72 53"01 2"07 2"18ca
2-75 2"65 22'05 11"90 4-85 54"19 1-61 2"34ca
2-60 2"31 23.17 9"12 6.14 54"59 2.07 2"08d
2'73 2"71 22.13 11"02 5'25 53"99 2"17 2"27cd
2-31 2"47 21'03 11-37 4-31 57"06 1'46 2'57c
16"09 23"27 22"29 19"68 30"66 6"02 44.34 10"30
aPercentage of the total peak area of the fatty acids listed.There were no significantdifferences in fatty acid percentages across fat quality grade. C.V., coefficientof variation. bMUFA, monounsaturated fatty acids (14:1 + 16: I + 18: I); SFA, saturated fatty acids (14:0+ 16:0+ 18:0). cdMeans in the same row with common superscripts do not differ (P > 0-05).
Fatty acid composition of Wagyu tissues
453
RESULTS
Fatty acid composition Experiment 1
Experiment 1 was designed to determine whether fatty acid compositional differences exist across Japanese fat quality grades. Mean percentages of fatty acids in s.c. adipose tissue samples showed no significant differences across fat quality grade (Table 1). Among fat quality grades, the only difference in fatty acid composition approaching significance was for myristate (C14:0), which tended (P < 0.07) to decrease with increasing fat quality grade. Commensurate with the decrease in palmitic acid seen with increasing fat quality grade was a general (though statistically insignificant) increase in oleic acid. The M U F D : S F A ratio ( 1 4 : 1 + 1 6 : 1 + 1 8 : 1 ) / (14:0 + 16:0 + 18:0) differed ( P < 0.05) among fat quality grades; quality grade 5 was significantly higher than grade 3. Experiment 2
In experiment 2, the fatty acid composition was compared among depot sites in crossbred Wagyu cattle raised in the USA (Table 2). Subcutaneous adipose tissue had significantly more myristoleate and palmitoleate, and significantly less stearate and linoleate than muscle or i.m. adipose tissue. M . l o n g i s s i m u s dorsi tissue had a higher percentage of palmitate as compared with marbling adipose tissue. There was no difference (P > 0.05) in the M U F A : S F A ratio across tissues. The M U F A : S F A ratio of the s.c. adipose tissue (1.46) was substantially less than the values observed in experiment 1. TABLE 2 Percentages o f M a j o r F a t t y Acids in 1/2-7/8 Crossbred W a g y u Steers (Experiment 2)
Fatty acid
14:0 14:1 16:0 16:1 18:0 18:1 18: 2 MUFA: SFA
Depot site
C.V.
Subcutaneous adipose tissue (n = 8)
Intramuscular adiposetissue (n = 8)
M. longissimus dorsi
3.78 2 "21a 28"59 ~b 8 "67a 7"68 b 47.12 1-95 b 1"46
3'41 1"23b 26"87 b 5"18 b 14-36 a 46-38 2.59 a 1"22
3'13 1"06b 30-31 a 5"35 b 11"07 b 46.34 2'73 a 1.19
(n = 8)
obM e a n s in the same row with c o m m o n superscripts d o n o t differ (P > 0"05).
16'23 30"48 8-90 19-01 21"01 8"98 20"99 18.97
C. A. Sturdivant, D. K. Lunt, G. C. Smith, S. B. Smith
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TABLE 3
Percentages of Major Fatty Acids in Tissues of Purebred Black Wagyu Cattle from Three Regions of Japan (Experiment 3) Fat O, acid
14:0 14:1 16:0 16 : 1 18:0 18:1 18:2 M U F A :SFA
Region
C.V.
Kagoshima (n = 15a)
Miyazaki (n = 12)
Gunma (n = 6)
2"29c 1'43 23'84 6'68 c 8"67b 54"13 b 2-66 1"82c
3"19b 1'43 25'14 7" 17c 8"27b 51"35 c 3-45 l'6Y
1'91 c 1"53 23"57 8"66b 6"01c 55"50 b 2"82 2"10b
12-14 27-44 6"23 16'86 20'33 4"80 39"21 12'39
a N u m b e r s in parentheses indicate total n u m b e r s o f s.c. and i.m. adipose tissues and M.
hmgissimus dorsi sampled for each region. D a t a are grouped across tissues.
hcMeans in the same row with common superscripts do not differ (P > 0-05). Experiment 3 There was no interaction (P > 0"10) between region and tissue site. For this reason, data for the experiment are presented as the main effects means for tissue and region in Japan. Of the regions in Japan evaluated, samples from the Gunma region had the lowest percentages of myristate and stearate (Table 3). Samples from this region also had the highest percentage of palmitoleate and had a greater percentage of oleate than samples from the Miyazaki region. This contributed to the higher M U F A : S F A ratio of the Gunma region (over 2-0). Tissues from the Miyazaki region had a significantly lower percentage of oleate as compared with the Gunma and Kagoshima regions, but these samples also had the most myristate. By depot site (Table 4), s.c. adipose tissue had the lowest percentage of myristic and palmitic acids, and the highest M U F A :SFA ratio of the depots examined. Muscle tissue was significantly higher in myristic and palmitic acids, and lower in oleic acid than the other tissues (Table 4).
DISCUSSION Increasing the concentration of oleate in meat could influence both palatability and perception of meat. Dryden & Marchello (1970) reported that M. longissimus dorsi with high percentages of oleate generally scored higher in taste panel evaluations. These results supported Waldman et al.
Fatty acid composition of Wagyu tissues
455
TABLE 4
Percentages of Major Fatty Acids in Tissues of Purebred Black Wagyu Cattle from Three Regions of Japan, Grouped by Tissue Site (Experiment 3) Fato, acid
Depot site Subcutaneous adipose tissue (n= 11")
14:0 14:1 16:0 16:1 18:0 18:1
18: 2 MUFA :SFA
2.35c 1.64 22'30a 7-52 8"32 55.19b 2.64 1-98b
Intramuscular adipose tissue (n= 11)
2-57bc 1'37 23"84c 6"62 8.71 54.10b 2.62 1.78c
C.V.
M. longissimus dorsi (n= 11) 2.73b 1.33 26.64b 7.53 7.19 50.81c 3-67 1-66c
12"14 27.44 6.23 16"86 20.33 4.80 39.21 12.39
"Numbers in parentheses indicate total numbers of s.c. and i.m. adipose tissues and M. longissimus dorsi sampled pooled across regions in Japan. pcMeans in the same row with common superscripts do not differ (P < 0'05). (1965), who reported that juiciness ratings for the M . longissimus dorsi were negatively correlated with contents o f myristate and palmitate, and positively correlated with the ratio of unsaturated :saturated fatty acids. The perception o f beef also m a y be improved by increasing its oleate content. It is generally accepted that the consumption o f oleate by humans reduces, or does not increase, serum low-density lipoprotein-cholesterol. F o r this reason, G r u n d y (1989) recommended that dietary fatty acids such as palmitic be replaced with oleic. Recently, K u b e n a & Rhee (1991) demonstrated that pork modified to contain a greater percentage of oleate (approximately 54% o f the total fatty acids) lowered low-density lipoprotein-cholesterol when fed to guinea-pigs. Thus, beef containing a greater concentration of oleate, and less palmitate, m a y be perceived as a more beneficial c o m p o n e n t of a diet designed to lower serum cholesterol. In anticipation o f liberalization o f the beef trade with the USA, the Japanese government revised beef carcass grading specifications in 1988. The new standards are much less stringent regarding the a m o u n t of marbling required to qualify for the highest quality grade. Ribbing was standardized at the 6th-7th rib interface. The grading system also was changed to be like the U S A system in that a yield and a quality grade are applied. N o uncoupling o f grades is allowed; b o t h a yield grade and a quality grade are assigned. Five quality grades are applied to beef carcasses in Japan: 1, 2, 3, 4 and 5,
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where 1 is the lowest and 5 is the highest or most desirable grade. Quality grades are determined by four factors: marbling score; color and brightness of lean; firmness and texture of lean; and color, luster and quality of fat (Anonymous, 1988). Quality of fat is in part determined by the firmness of the fat, in that a softer fat is considered more desirable. We hypothesized that differences in fat texture, and hence quality, were caused by differences in fatty acid composition. There were no differences in the individual fatty acids across fat quality grades, although the highest fat quality grade exhibited the greatest monounsaturated:saturated fatty acid ratio. Thus, the basis for differences in fat quality grade remains to be demonstrated. The s.c. adipose tissue samples from the purebred Black Wagyu cattle of five Japanese fat quality grades exhibited extraordinarily high percentages of all of the monounsaturated fatty acids. The concentrations of myristoleate, palmitoleate and oleate exceeded any that we had reported previously for steers, heifers or cows (St. John et al., 1987, 1991; Sweeten et al., 1990; Huerta-Leidenz et al., 1991, in press). The elevated monosaturated fatty acids in the adipose tissue of the Wagyu cattle could have been the result of increased absorption of the fatty acids (i.e. absorbed dietary monounsaturated fatty acids), or elevated activity of stearoyl-CoA desaturase within adipose tissue. Influences of absorbed monounsaturated fatty acids are unlikely. Myristoleate and palmitoleate are not found in abundance in feeds, and those that are ingested would be largely hydrogenated to the saturated counterparts by ruminal microflora (Smith, 1991). Bovine adipose tissue contains an active stearoyl-CoA desaturase, which greatly exceeds activity observed in liver (St. John et al., 1991; Smith, 1991). Stearoyl-CoA desaturase is a A - 9 desaturase, which converts stearate, palmitate and myristate to their corresponding n - 9 monounsaturated fatty acids. Although bovine muscle and intestinal luminal epithelial tissue also exhibit stearoyl-CoA desaturase activity (Smith, 1991) fatty acid desaturation by the stearoyl-CoA desaturase is quantitatively most important in adipose tissue. Because stearoyl-CoA desaturase catalyzes the conversion of all saturated fatty acids to n - 9 monounsaturated fatty acids, a single enzyme could be responsible for the elevated monounsaturated fatty acids observed in Wagyu adipose tissue. If elevated stearoyl-CoA desaturase activity is responsible for the elevated monounsaturated fatty acids observed in experiment 1 and reported by Tanaka (1985), then this would support a genetic basis for this unusual fatty acid composition. However, the results of experiment 2 would argue against any genetic predisposition of Wagyu cattle to synthesize and deposit monounsaturated fatty acids in their tissues. The monounsaturated fatty
Fatty acid composition of Wagyu tissues
457
acid values reported for M. longissimus dorsi and s.c. and i.m. adipose tissue for the Wagyu x Angus crossbred cattle were essentially identical to those reported for purebred Angus steers (Sweeten et aL, 1990), raised under typical US feedlot conditions. However, the crossbred Wagyu cattle were derived from crosses of Black and Red Wagyu bulls on Angus heifers. The data for experiment 1, and those reported by Tanaka (1985) were derived from samples from purebred Black Wagyu cattle. The contribution of the Red Wagyu and Angus genetics may have diluted the contribution of the Black Wagyu cattle. The region in Japan from which the samples were obtained also could influence the fatty acid composition of the tissues. Regions (prefectures) within Japan have been rigorously characterized for their Wagyu sire history (Namikawa, date unknown). This could have resulted in distinct genetic pools regulating fatty acid composition of the tissues. The results of experiment 3 suggest that this is the case. Although based on a small number of samples, the MUFA:SFA ratio of s.c. adipose tissue from the Gunma prefecture (2:10) was equivalent to values reported for samples from the Japanese purebred cattle used in experiment 1, and was significantly greater than the MUFA :SFA ratios for samples from the Kagoshima and Miyazaki prefectures. In summary, this investigation supports the observations of Tanaka (1985), in that purebred Wagyu cattle from Japan displayed elevated monounsaturated fatty acids in their adipose tissues. Our results suggest a genetic basis for this compositional difference, as do the data of Tanaka (1985). However, because the production of these cattle has not been characterized fully, environmental influences on tissue fatty acid composition cannot be ruled out. Because of the high marbling content of meat from Wagyu cattle, the elevated concentration of oleate in Wagyu beef has no practical health significance. However, if the genes dictating the compositional differences in Wagyu beef can be incorporated into a lean beef program, then there is potential for producing beef with increased palatability and healthfulness.
ACKNOWLEDGEMENTS Technical article 30032 from the Texas Agricultural Experiment Station. Adipose tissue samples for experiment 1 were generously provided by Itoham Foods, Nishinomiya, Japan. Samples for experiment 3 were obtained with the assistance of the Japan America Friendship Foundation, Gardena, CA, USA.
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REFERENCES Anonymous (1988). New Beef Carcass Grading Standards. Japan Meat Grading Association, Tokyo. Dryden, F. D. & Marchello, J. A. (1970). J. Anim. Sci., 31, 36. Eichorn, J. M., Coleman, L. J., Wakayama, E. J., Blomquist, G. J., Bailey, C. M. & Jenkins, T. G. (1986). J. Anim. Sci., 63, 781. Grundy, S. M. (1989). J. Nutr., 119, 529. Huerta-Leindenz, N. O., Cross, H. R., Lunt, D. K., Pelton, L. S., Savell, J. W. & Smith, S. B. (1991). J. Anita. Sci., 69, 3665. Huerta-Leindenz, N. O., Cross, H. R., Savell, J. W., Lunt, D. K., Baker, J. F., Pelton, L. S. & Smith, S. B. (in press). J. Anita. Sci. Kubena, K. & Rhee, K. S. (1991). INFORM, 2, 364. Morrison, W. R. & Smith, L. M. (1964). J. Lipid Res., 5, 600. Namikawa, K. (date unknown). Historical breeding processes of Japanese beef cattle and preservation of genetic resources as economic farm animals. Wagyu Cattle Registry Association, Kyoto University, Kyoto, Japan. Ott, L. (1984). An Introduction to Statistical Methods and Data Analysis, 2nd edn. PWS, Boston, MA. SAS (1986). S A S User's Guide: Statistics. SAS Institute, Inc., Cary, NC. St. John, L. C., Young, C. R., Knabe, D. A., Schelling, G. T., Grundy, S. M. & Smith, S. B. (1987). J. Anita. Sci., 64, 1441. St. John, L. C., Lunt, D. K. & Smith, S. B. (1991). J. Anita. Sci., 69, 1064. Smith, S. B. (1991). In Fat and Cholesterol Reduced Foods. Technologies and Strategies, eds. C. Haberstroh & C. E. Morris. Gulf, Houston, TX, p. 75. Sweeten, M. K., Cross, H. R., Smith, G. C. & Smith, S. B. (1990). J. FoodSci., 55, 43. Tanaka, S. (1985). Jap. J. Dairy Food Sci., 34, 92. Waldman, R. C., Suess, G. G., Lewis, R. W., Bray, R. W. & Brungardt, V. H. (1965). J. Anim. Sci., 24, 869. Yoshimura, T. & Namikawa, K. (1983). Jpn. J. Zootech. Sci., 54, 97.
Fatty Acid Composition of Subcutaneous and Intramuscular Adipose Tissues and M. longissimus dorsi of Wagyu Cattle C. A. Sturdivant, D. K. Lunt, G. C. Smith & S. B. Smith* Department of Animal Science, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843, USA (Received 30 June 1991; revised version received 22 November 1991; accepted 24 November 1991) ABSTRACT Three experiments were conducted to document the fatty acid composition of tissues from purebred Wagyu cattle from Japan and North American crossbred Wagyu. In experiment 1, subcutaneous (s.c.) adipose tissues ( n = 23) were obtained from Japanese cattle representing the.17veJapanese fat quality grades. The monounsaturated:saturated fotty acid ratio (MUFA:SFA ) was greatest in.fat quality grade 5 samples (2.5 7) and least in thefat quality grade 3 samples (2.08: P < 0"05). In experiment 2, M. longissimus dorsi and the associated intramuscular ( i.m.) and s.c. adipose tissues were obtahled /)'om carcasses of Wagyu crossbred steers (1/2-7/8) raised in the USA. Fatty acid composition varied among depots, but the MUFA:SFA ratio in s.c. adipose tissue (1"46) was not d(fferent from values reported for other breeds' o['cattlc. In experiment 3, samples of M. longissimus dorsi ribsteaks were obtained from three regions in Japan. Samples from the Gunma region had the greatest ( P < 0"05) M UFA :SFA ratio (2"10), relative to samples /i'~m the Kagoshima (1.82) and Miyazaki (1"65) regions. The data indicate that bee/ from purebred Wagyu cattle raised in Japan is enriched in monounsaturated fatty acids, and that the degree of enrichment depends upon the region of Japan from which the samples were obtained. INTRODUCTION Because o f the difficulties in modifying the fatty acid composition of bovine tissues by dietary means (e.g. Smith, 1991), selection of cattle with a greater * To whom correspondence should be addressed. 449 Meat Science0309-1740/92/$05.00 © 1992 ElsevierScience Publishers Ltd, England. Printed in Great Britain
450
C. A. Sturdivant, D. K. Lunt, G. C. Smith, S. B. Smith
genetic propensity to deposit specific fatty acids may be the primary means by which fatty acid composition is modified in ruminant species. Breed comparisons of fatty acid composition are difficult because of the confounding of age, plane of nutrition and other extrinsic factors (Yoshimura & Namikawa, 1983; Eichorn et al., 1986). In a recent investigation in which these factors were controlled, s.c. adipose tissue from mature Bos indicus cows contained greater percentages of the monounsaturated fatty acids myristoleate (14:1), palmitoleate (16:1) and oleate (18:1) than the adipose tissue from Bos taurus cows (Huerta-Leidenz et al., in press). This resulted in a greater ratio of monounsaturated:saturated (MUFA: SFA) fatty acids in adipose tissue from the Bos indicus cows (1.85 vs 1.54 for the Bos taurus cows). Although the differences in the individual fatty acids were not great (e.g. 49-6% oleate in Bos indicus adipose tissue vs 47.7% oleate in the Bos taurus adipose tissue), the results of Huerta-Leidenz et al. (in press) suggested that genetic differences do exist in the ability of cattle to synthesize and deposit monounsaturated fatty acids. Tanaka (1985) reported that s.c. adipose tissue from Japanese Black (Wagyu) steers contained over 48% oleate and had an M U F A :SFA ratio of approximately 1.5, compared with an M U F A : S F A ratio of 1.2 for adipose tissue from Japanese Shorthorn and Holstein steers. The objectives of this research were to provide additional information about the genetic basis for differences in fatty acid composition among breeds of cattle, and to further characterize the Wagyu breed, first by documenting fatty acid compositional differences among Japanese fat quality grades. The initial interest in evaluating differences among fat quality grades was generated by the subjective observation that increases in fat quality grade (which are ranked from 1 to 5) were associated with softer s.c. adipose tissue on chilled carcasses. The extraordinarily high concentrations of monounsaturated fatty acids in s.c. adipose tissue from purebred Black Wagyu cattle from Japan led to investigations of tissues from crossbred Wagyu steers raised in the USA. This was followed by investigations of regional differences in fatty acid composition.
MATERIALS AND METHODS
Experimental design Experiment 1
Twenty-three samples of s.c. adipose tissue were obtained from a commercial meat processing plant in Japan and transported on dry ice to the Department of Animal Science at Texas A & M University. Samples were
Fatty acid composition of Wagyu tissues
451
obtained at slaughter from Japanese Black Wagyu steers representing the five Japanese fat quality grades. Japanese Black Wagyu steers typically are slaughtered at 30-34 months of age. Samples were held frozen at - 2 0 ° C until analyzed for fatty acid composition.
Experiment 2 One-half to 7/8 Wagyu crossbred steers (n = 8) were fed a corn-milo feedlot ration free choice from a covered self-feeder for 174 days and then fed a corn-based feedlot ration until time of slaughter. Both Black and Red Wagyu bulls were mated to Angus and Hereford × Angus cows to produce the steers sampled in this experiment. Cattle were slaughtered when backfat thickness reached 1"8 cm, as determined by ultrasonography. The steers were approximately 27 months of age at slaughter. After the carcasses had been fabricated, samples of s.c. adipose tissue, M. longissimus dorsi and i.m. (marbling) adipose tissue from within the muscle were obtained by dissection. Samples were held frozen at - 2 0 ° C until analyzed for fatty acid composition. Experiment 3 The USA recently has allowed importation of Japanese beef. Eleven strip steaks from Japanese Black Wagyu steers produced and slaughtered in Japan were obtained from outlets in the USA. The steaks came from cattle representing three different regions of Japan: the Kagoshima region (n = 5), the Miyazaki region (n = 4) and the Gunma region (n = 2). From each steak, s.c. adipose tissue, M. longissimus dorsi, and i.m. adipose tissue from within the muscle were obtained by dissection. For subsequent analyses, the raw s.c. adipose tissue, muscle, and marbling samples were stored at -20°C.
Fatty acid analysis Fatty acids were determined by gas chromatography as described by HuertaLeidenz et al. (1991). An aliquot of the total lipid extract from each of the samples was freed of solvent under nitrogen using an analytical evaporator (Meyer N-Evap, Organomation Associates Inc., Berlin, MA). Each volume of the aliquot was calculated to yield approximately 25 mg of total lipid extract from the longissimus dorsi muscle. The extract was methylated with boron trifluoride-methanol following the procedure described by Morrison & Smith (1964). Fatty acid methyl esters (FAME) were analyzed using a flame ionization gas chromatograph (Packard, Model 437A, Chrompack Inc., Raritan, N J) equipped with a 2 m × 3"175 mm stainless steel column packed with 15% cyanopropylphenylpolysiloxane (9:1w/w) on a 100-120 mesh solid support. The column was run isothermally at a temperature of 180°C.
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F A M E in 1/A o f h e x a n e was delivered into the column using a microsyringe. The injection port and detector were maintained at 250°C. Flow rates were 16ml/min for the carrier gas (nitrogen), 34ml/min for hydrogen, and 214ml/min for breathing air. C h r o m a t o g r a m s were recorded with a computing integrator (Spectraphysics Model SP4290, San Jose, CA). The gas c h r o m a t o g r a p h system was calibrated with standard F A M E mixtures (Nu-Chek Prep. Inc., Elysian, MN). Identification o f sample fatty acids was made by comparing the relative retention times of F A M E peaks from samples with those of standards.
Statistical analysis Data were analyzed with the General Linear Program (SAS, 1986). Experiments 1 and 2 were analyzed by one-way analysis o f variance (Ott, 1984). F o r experiment 1, the main effect tested was fat quality grade. F o r experiment 2, the main effect tested was depot site. Experiment 3 was analyzed as a 3 × 3 factorial design (region x tissue site). The main effects analyzed were depot site and regions within Japan, and the region x tissue site interaction was tested. The analysis o f variance was used to indicate significant differences (P < 0"05) a m o n g means. When indicated by analysis o f variance, means were separated by the S t u d e n t - N e w m a n - K e u l s method (Ott, 1984).
TABLE 1 Percentages of Major Fatty Acids in Subcutaneous Adipose Tissue Samples of Wagyu Cattle as Affected by Japanese Fat Quality Grade (Experiment 1)a Fatty acid
14:0 14:1 16:0 16:1 18:0 18:1 18:2 MUFA:SFAb
Quality grade
C.V.
1 (n=5)
2 (n=4)
3 (n=4)
4 (n=6)
5 (n=4)
3"19 2"70 22.99 11'32 4'72 53"01 2"07 2"18ca
2-75 2"65 22'05 11"90 4-85 54"19 1-61 2"34ca
2-60 2"31 23.17 9"12 6.14 54"59 2.07 2"08d
2'73 2"71 22.13 11"02 5'25 53"99 2"17 2"27cd
2-31 2"47 21'03 11-37 4-31 57"06 1'46 2'57c
16"09 23"27 22"29 19"68 30"66 6"02 44.34 10"30
aPercentage of the total peak area of the fatty acids listed.There were no significantdifferences in fatty acid percentages across fat quality grade. C.V., coefficientof variation. bMUFA, monounsaturated fatty acids (14:1 + 16: I + 18: I); SFA, saturated fatty acids (14:0+ 16:0+ 18:0). cdMeans in the same row with common superscripts do not differ (P > 0-05).
Fatty acid composition of Wagyu tissues
453
RESULTS
Fatty acid composition Experiment 1
Experiment 1 was designed to determine whether fatty acid compositional differences exist across Japanese fat quality grades. Mean percentages of fatty acids in s.c. adipose tissue samples showed no significant differences across fat quality grade (Table 1). Among fat quality grades, the only difference in fatty acid composition approaching significance was for myristate (C14:0), which tended (P < 0.07) to decrease with increasing fat quality grade. Commensurate with the decrease in palmitic acid seen with increasing fat quality grade was a general (though statistically insignificant) increase in oleic acid. The M U F D : S F A ratio ( 1 4 : 1 + 1 6 : 1 + 1 8 : 1 ) / (14:0 + 16:0 + 18:0) differed ( P < 0.05) among fat quality grades; quality grade 5 was significantly higher than grade 3. Experiment 2
In experiment 2, the fatty acid composition was compared among depot sites in crossbred Wagyu cattle raised in the USA (Table 2). Subcutaneous adipose tissue had significantly more myristoleate and palmitoleate, and significantly less stearate and linoleate than muscle or i.m. adipose tissue. M . l o n g i s s i m u s dorsi tissue had a higher percentage of palmitate as compared with marbling adipose tissue. There was no difference (P > 0.05) in the M U F A : S F A ratio across tissues. The M U F A : S F A ratio of the s.c. adipose tissue (1.46) was substantially less than the values observed in experiment 1. TABLE 2 Percentages o f M a j o r F a t t y Acids in 1/2-7/8 Crossbred W a g y u Steers (Experiment 2)
Fatty acid
14:0 14:1 16:0 16:1 18:0 18:1 18: 2 MUFA: SFA
Depot site
C.V.
Subcutaneous adipose tissue (n = 8)
Intramuscular adiposetissue (n = 8)
M. longissimus dorsi
3.78 2 "21a 28"59 ~b 8 "67a 7"68 b 47.12 1-95 b 1"46
3'41 1"23b 26"87 b 5"18 b 14-36 a 46-38 2.59 a 1"22
3'13 1"06b 30-31 a 5"35 b 11"07 b 46.34 2'73 a 1.19
(n = 8)
obM e a n s in the same row with c o m m o n superscripts d o n o t differ (P > 0"05).
16'23 30"48 8-90 19-01 21"01 8"98 20"99 18.97
C. A. Sturdivant, D. K. Lunt, G. C. Smith, S. B. Smith
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TABLE 3
Percentages of Major Fatty Acids in Tissues of Purebred Black Wagyu Cattle from Three Regions of Japan (Experiment 3) Fat O, acid
14:0 14:1 16:0 16 : 1 18:0 18:1 18:2 M U F A :SFA
Region
C.V.
Kagoshima (n = 15a)
Miyazaki (n = 12)
Gunma (n = 6)
2"29c 1'43 23'84 6'68 c 8"67b 54"13 b 2-66 1"82c
3"19b 1'43 25'14 7" 17c 8"27b 51"35 c 3-45 l'6Y
1'91 c 1"53 23"57 8"66b 6"01c 55"50 b 2"82 2"10b
12-14 27-44 6"23 16'86 20'33 4"80 39"21 12'39
a N u m b e r s in parentheses indicate total n u m b e r s o f s.c. and i.m. adipose tissues and M.
hmgissimus dorsi sampled for each region. D a t a are grouped across tissues.
hcMeans in the same row with common superscripts do not differ (P > 0-05). Experiment 3 There was no interaction (P > 0"10) between region and tissue site. For this reason, data for the experiment are presented as the main effects means for tissue and region in Japan. Of the regions in Japan evaluated, samples from the Gunma region had the lowest percentages of myristate and stearate (Table 3). Samples from this region also had the highest percentage of palmitoleate and had a greater percentage of oleate than samples from the Miyazaki region. This contributed to the higher M U F A : S F A ratio of the Gunma region (over 2-0). Tissues from the Miyazaki region had a significantly lower percentage of oleate as compared with the Gunma and Kagoshima regions, but these samples also had the most myristate. By depot site (Table 4), s.c. adipose tissue had the lowest percentage of myristic and palmitic acids, and the highest M U F A :SFA ratio of the depots examined. Muscle tissue was significantly higher in myristic and palmitic acids, and lower in oleic acid than the other tissues (Table 4).
DISCUSSION Increasing the concentration of oleate in meat could influence both palatability and perception of meat. Dryden & Marchello (1970) reported that M. longissimus dorsi with high percentages of oleate generally scored higher in taste panel evaluations. These results supported Waldman et al.
Fatty acid composition of Wagyu tissues
455
TABLE 4
Percentages of Major Fatty Acids in Tissues of Purebred Black Wagyu Cattle from Three Regions of Japan, Grouped by Tissue Site (Experiment 3) Fato, acid
Depot site Subcutaneous adipose tissue (n= 11")
14:0 14:1 16:0 16:1 18:0 18:1
18: 2 MUFA :SFA
2.35c 1.64 22'30a 7-52 8"32 55.19b 2.64 1-98b
Intramuscular adipose tissue (n= 11)
2-57bc 1'37 23"84c 6"62 8.71 54.10b 2.62 1.78c
C.V.
M. longissimus dorsi (n= 11) 2.73b 1.33 26.64b 7.53 7.19 50.81c 3-67 1-66c
12"14 27.44 6.23 16"86 20.33 4.80 39.21 12.39
"Numbers in parentheses indicate total numbers of s.c. and i.m. adipose tissues and M. longissimus dorsi sampled pooled across regions in Japan. pcMeans in the same row with common superscripts do not differ (P < 0'05). (1965), who reported that juiciness ratings for the M . longissimus dorsi were negatively correlated with contents o f myristate and palmitate, and positively correlated with the ratio of unsaturated :saturated fatty acids. The perception o f beef also m a y be improved by increasing its oleate content. It is generally accepted that the consumption o f oleate by humans reduces, or does not increase, serum low-density lipoprotein-cholesterol. F o r this reason, G r u n d y (1989) recommended that dietary fatty acids such as palmitic be replaced with oleic. Recently, K u b e n a & Rhee (1991) demonstrated that pork modified to contain a greater percentage of oleate (approximately 54% o f the total fatty acids) lowered low-density lipoprotein-cholesterol when fed to guinea-pigs. Thus, beef containing a greater concentration of oleate, and less palmitate, m a y be perceived as a more beneficial c o m p o n e n t of a diet designed to lower serum cholesterol. In anticipation o f liberalization o f the beef trade with the USA, the Japanese government revised beef carcass grading specifications in 1988. The new standards are much less stringent regarding the a m o u n t of marbling required to qualify for the highest quality grade. Ribbing was standardized at the 6th-7th rib interface. The grading system also was changed to be like the U S A system in that a yield and a quality grade are applied. N o uncoupling o f grades is allowed; b o t h a yield grade and a quality grade are assigned. Five quality grades are applied to beef carcasses in Japan: 1, 2, 3, 4 and 5,
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where 1 is the lowest and 5 is the highest or most desirable grade. Quality grades are determined by four factors: marbling score; color and brightness of lean; firmness and texture of lean; and color, luster and quality of fat (Anonymous, 1988). Quality of fat is in part determined by the firmness of the fat, in that a softer fat is considered more desirable. We hypothesized that differences in fat texture, and hence quality, were caused by differences in fatty acid composition. There were no differences in the individual fatty acids across fat quality grades, although the highest fat quality grade exhibited the greatest monounsaturated:saturated fatty acid ratio. Thus, the basis for differences in fat quality grade remains to be demonstrated. The s.c. adipose tissue samples from the purebred Black Wagyu cattle of five Japanese fat quality grades exhibited extraordinarily high percentages of all of the monounsaturated fatty acids. The concentrations of myristoleate, palmitoleate and oleate exceeded any that we had reported previously for steers, heifers or cows (St. John et al., 1987, 1991; Sweeten et al., 1990; Huerta-Leidenz et al., 1991, in press). The elevated monosaturated fatty acids in the adipose tissue of the Wagyu cattle could have been the result of increased absorption of the fatty acids (i.e. absorbed dietary monounsaturated fatty acids), or elevated activity of stearoyl-CoA desaturase within adipose tissue. Influences of absorbed monounsaturated fatty acids are unlikely. Myristoleate and palmitoleate are not found in abundance in feeds, and those that are ingested would be largely hydrogenated to the saturated counterparts by ruminal microflora (Smith, 1991). Bovine adipose tissue contains an active stearoyl-CoA desaturase, which greatly exceeds activity observed in liver (St. John et al., 1991; Smith, 1991). Stearoyl-CoA desaturase is a A - 9 desaturase, which converts stearate, palmitate and myristate to their corresponding n - 9 monounsaturated fatty acids. Although bovine muscle and intestinal luminal epithelial tissue also exhibit stearoyl-CoA desaturase activity (Smith, 1991) fatty acid desaturation by the stearoyl-CoA desaturase is quantitatively most important in adipose tissue. Because stearoyl-CoA desaturase catalyzes the conversion of all saturated fatty acids to n - 9 monounsaturated fatty acids, a single enzyme could be responsible for the elevated monounsaturated fatty acids observed in Wagyu adipose tissue. If elevated stearoyl-CoA desaturase activity is responsible for the elevated monounsaturated fatty acids observed in experiment 1 and reported by Tanaka (1985), then this would support a genetic basis for this unusual fatty acid composition. However, the results of experiment 2 would argue against any genetic predisposition of Wagyu cattle to synthesize and deposit monounsaturated fatty acids in their tissues. The monounsaturated fatty
Fatty acid composition of Wagyu tissues
457
acid values reported for M. longissimus dorsi and s.c. and i.m. adipose tissue for the Wagyu x Angus crossbred cattle were essentially identical to those reported for purebred Angus steers (Sweeten et aL, 1990), raised under typical US feedlot conditions. However, the crossbred Wagyu cattle were derived from crosses of Black and Red Wagyu bulls on Angus heifers. The data for experiment 1, and those reported by Tanaka (1985) were derived from samples from purebred Black Wagyu cattle. The contribution of the Red Wagyu and Angus genetics may have diluted the contribution of the Black Wagyu cattle. The region in Japan from which the samples were obtained also could influence the fatty acid composition of the tissues. Regions (prefectures) within Japan have been rigorously characterized for their Wagyu sire history (Namikawa, date unknown). This could have resulted in distinct genetic pools regulating fatty acid composition of the tissues. The results of experiment 3 suggest that this is the case. Although based on a small number of samples, the MUFA:SFA ratio of s.c. adipose tissue from the Gunma prefecture (2:10) was equivalent to values reported for samples from the Japanese purebred cattle used in experiment 1, and was significantly greater than the MUFA :SFA ratios for samples from the Kagoshima and Miyazaki prefectures. In summary, this investigation supports the observations of Tanaka (1985), in that purebred Wagyu cattle from Japan displayed elevated monounsaturated fatty acids in their adipose tissues. Our results suggest a genetic basis for this compositional difference, as do the data of Tanaka (1985). However, because the production of these cattle has not been characterized fully, environmental influences on tissue fatty acid composition cannot be ruled out. Because of the high marbling content of meat from Wagyu cattle, the elevated concentration of oleate in Wagyu beef has no practical health significance. However, if the genes dictating the compositional differences in Wagyu beef can be incorporated into a lean beef program, then there is potential for producing beef with increased palatability and healthfulness.
ACKNOWLEDGEMENTS Technical article 30032 from the Texas Agricultural Experiment Station. Adipose tissue samples for experiment 1 were generously provided by Itoham Foods, Nishinomiya, Japan. Samples for experiment 3 were obtained with the assistance of the Japan America Friendship Foundation, Gardena, CA, USA.
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REFERENCES Anonymous (1988). New Beef Carcass Grading Standards. Japan Meat Grading Association, Tokyo. Dryden, F. D. & Marchello, J. A. (1970). J. Anim. Sci., 31, 36. Eichorn, J. M., Coleman, L. J., Wakayama, E. J., Blomquist, G. J., Bailey, C. M. & Jenkins, T. G. (1986). J. Anim. Sci., 63, 781. Grundy, S. M. (1989). J. Nutr., 119, 529. Huerta-Leindenz, N. O., Cross, H. R., Lunt, D. K., Pelton, L. S., Savell, J. W. & Smith, S. B. (1991). J. Anita. Sci., 69, 3665. Huerta-Leindenz, N. O., Cross, H. R., Savell, J. W., Lunt, D. K., Baker, J. F., Pelton, L. S. & Smith, S. B. (in press). J. Anita. Sci. Kubena, K. & Rhee, K. S. (1991). INFORM, 2, 364. Morrison, W. R. & Smith, L. M. (1964). J. Lipid Res., 5, 600. Namikawa, K. (date unknown). Historical breeding processes of Japanese beef cattle and preservation of genetic resources as economic farm animals. Wagyu Cattle Registry Association, Kyoto University, Kyoto, Japan. Ott, L. (1984). An Introduction to Statistical Methods and Data Analysis, 2nd edn. PWS, Boston, MA. SAS (1986). S A S User's Guide: Statistics. SAS Institute, Inc., Cary, NC. St. John, L. C., Young, C. R., Knabe, D. A., Schelling, G. T., Grundy, S. M. & Smith, S. B. (1987). J. Anita. Sci., 64, 1441. St. John, L. C., Lunt, D. K. & Smith, S. B. (1991). J. Anita. Sci., 69, 1064. Smith, S. B. (1991). In Fat and Cholesterol Reduced Foods. Technologies and Strategies, eds. C. Haberstroh & C. E. Morris. Gulf, Houston, TX, p. 75. Sweeten, M. K., Cross, H. R., Smith, G. C. & Smith, S. B. (1990). J. FoodSci., 55, 43. Tanaka, S. (1985). Jap. J. Dairy Food Sci., 34, 92. Waldman, R. C., Suess, G. G., Lewis, R. W., Bray, R. W. & Brungardt, V. H. (1965). J. Anim. Sci., 24, 869. Yoshimura, T. & Namikawa, K. (1983). Jpn. J. Zootech. Sci., 54, 97.